The field of metabolomics seeks to characterize the suite of small molecules that comprise the endproducts of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization-liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene-divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from < 1 pM to 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.Metabolomics is an "omics" technique that seeks to measure the small organic biomolecules produced by cells (Oliver et al. 1998;Fiehn 2002). Because these small molecules are the end-products of multiple levels of metabolic regulation, their concentrations provide a temporal snapshot of the metabolic state or phenotype of an organism. In particular, metabolites produced by nonenzymatic reactions, such as those formed by reaction with a radical oxygen species, or whose production is regulated by other small molecules, must be monitored directly because their production cannot be inferred from genomic or proteomic information. Metabolomics can be used as a diagnostic tool, identifying biomarkers of disease within the human metabolome, such as cancers (Armitage and Barbas 2014) and Crohn's Disease (Jansson et al. 2009). Metabolomics has also been applied in a wide range of organisms and environments, examining how metabolite abundances respond to environmental factors. In the oceans, marine metabolites have been a valuable source of new natural products, while other metabolomics applications are still rare but growing. For example, recent marine culture experiments have revealed metabolite production not predicted by genomic information (Baran et al. 2010;Fiore et al. 2015), metabolic shifts in response to a specific metabolite (Johnson et al. 2016), and changes in the quantity and composition of metabolite production during coculturing (Paul et al. 2012). Complementary field ...
Microbes, the foundation of the marine foodweb, do not function in isolation, but rather rely on molecular level interactions among species to thrive. Although certain types of interactions between autotrophic and heterotrophic microorganisms have been well documented, the role of specific organic molecules in regulating inter-species relationships and supporting growth are only beginning to be understood. Here, we examine one such interaction by characterizing the metabolic response of a heterotrophic marine bacterium, Ruegeria pomeroyi DSS-3, to growth on dimethylsulfoniopropionate (DMSP), an abundant organosulfur metabolite produced by phytoplankton. When cultivated on DMSP, R. pomeroyi synthesized a quorum-sensing molecule, N-(3-oxotetradecanoyl)-l-homoserine lactone, at significantly higher levels than during growth on propionate. Concomitant with the production of a quorum-sensing molecule, we observed differential production of intra- and extracellular metabolites including glutamine, vitamin B2 and biosynthetic intermediates of cyclic amino acids. Our metabolomics data indicate that R. pomeroyi changes regulation of its biochemical pathways in a manner that is adaptive for a cooperative lifestyle in the presence of DMSP, in anticipation of phytoplankton-derived nutrients and higher microbial density. This behavior is likely to occur on sinking marine particles, indicating that this response may impact the fate of organic matter.
Microbial metabolism plays a primary role in shaping the marine carbon cycle through processes of carbon fixation and remineralization. Many metabolic intermediates pass through the reservoir of marine dissolved organic matter (DOM), as compounds move among microbes as part of complex ecological networks of interactions. Environmental metabolomics can be used to identify and quantify these compounds, and thus will provide insight into the chemical underpinnings of microbial networks at the foundation of global biogeochemical cycles. Here we present methods for metabolite profiling (untargeted metabolomics) and for relative quantification (targeted metabolomics) of intracellular and extracellular metabolites from marine microbes. We describe our approach to method development with regard to metabolite extraction and instrumental analysis, culminating in the methods currently in use in our laboratory.
Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca 2؉ ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca
Marine sinking particles transport carbon from the surface and bury it in deep‐sea sediments, where it can be sequestered on geologic time scales. The combination of the surface ocean food web that produces these particles and the particle‐associated microbial community that degrades them creates a complex set of variables that control organic matter cycling. We use targeted metabolomics to characterize a suite of small biomolecules, or metabolites, in sinking particles and compare their metabolite composition to that of the suspended particles in the euphotic zone from which they are likely derived. These samples were collected in the South Atlantic subtropical gyre, as well as in the equatorial Atlantic region and the Amazon River plume. The composition of targeted metabolites in the sinking particles was relatively similar throughout the transect, despite the distinct oceanic regions in which they were generated. Metabolites possibly derived from the degradation of nucleic acids and lipids, such as xanthine and glycine betaine, were an increased mole fraction of the targeted metabolites in the sinking particles relative to surface suspended particles, while algal‐derived metabolites like the osmolyte dimethylsulfoniopropionate were a smaller fraction of the observed metabolites on the sinking particles. These compositional changes are shaped both by the removal of metabolites associated with detritus delivered from the surface ocean and by production of metabolites by the sinking particle‐associated microbial communities. Furthermore, they provide a basis for examining the types and quantities of metabolites that may be delivered to the deep sea by sinking particles.
Marine eukaryotic phytoplankton adapt to low phosphorus (P) in the oceans through a variety of step-wise mechanisms including lipid substitution and decreased nucleic acid content. Here, we examined the impact of low P concentrations on intracellular metabolites whose abundances can be quickly adjusted by cellular regulation within laboratory cultures of three model phytoplankton and in field samples from the Atlantic and Pacific Oceans. We quantified the relative abundances of monophosphate nucleotides and their corresponding nucleosides, using a combination of targeted and untargeted metabolomics methods. Under P-deficient conditions, we observed a marked decrease in adenosine 5 0 -monophosphate (AMP) with a concomitant increase in adenosine. This shift occurred within all detected pairs of monophosphate nucleotides and nucleosides, and was consistent with previous work showing transcriptional changes in nucleotide synthesis and salvage under P-deficient conditions for model eukaryotes. In the field, we observed AMP-to-adenosine ratios that were similar to those in laboratory culture under P-deficient conditions. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Scientific Significance StatementPhosphorus (P) is a central element in cellular metabolism that can limit primary production of eukaryotic phytoplankton in the ocean. Adaptation to low P is known to drive metabolic restructuring in eukaryotic phytoplankton, but the specific adaptive responses of cellular metabolites are poorly understood. Here we show that three model phytoplankton alter their metabolites under P deficiency, relative to P-replete conditions. We present evidence for a new model of P allocation within cells, where monophosphate nucleotides can act as a flexible P storage pool, allowing rapid and dynamic distribution of P to cellular processes.
Global-scale surveys of plankton communities using “omics” techniques have revolutionized our understanding of the ocean. Lipidomics has demonstrated the potential to add further essential insights on ocean ecosystem function but has yet to be applied on a global scale. We analyzed 930 lipid samples across the global ocean using a uniform high-resolution accurate-mass mass spectrometry analytical workflow, revealing previously unknown characteristics of ocean planktonic lipidomes. Focusing on 10 molecularly diverse glycerolipid classes, we identified 1151 distinct lipid species, finding that fatty acid unsaturation (i.e., number of carbon-carbon double bonds) is fundamentally constrained by temperature. We predict substantial declines in the essential fatty acid eicosapentaenoic acid over the next century, which are likely to have serious deleterious effects on economically critical fisheries.
The production and consumption of organic matter by marine organisms plays a central role in the marine carbon cycle. Labile organic compounds (metabolites) are the major currency of energetic demands and organismal interaction, but these compounds remain elusive because of their rapid turnover and concomitant minuscule concentrations in the dissolved organic matter pool. Organic osmolytes are a group of small metabolites synthesized at high intracellular concentrations (mM) to regulate cellular osmolarity and have the potential to be released as abundant dissolved substrates. Osmolytes may represent an essential currency of exchange among heterotrophic prokaryotes and primary and secondary producers in marine food webs. For example, the well-known metabolite dimethylsulfoniopropionate (DMSP) is used as an osmolyte by some phytoplankton and can be subsequently metabolized by 60% of the marine bacterial community, supplying up to 13% of the bacterial carbon demand and 100% of the bacterial sulfur demand. While marine osmolytes have been studied for decades, our understanding of their cycling and significance within microbial communities is still far from comprehensive. Here, we surveyed the genes responsible for synthesis, breakdown, and transport of 14 key osmolytes. We systematically searched for these genes across marine bacterial genomes (n = 897) and protistan transcriptomes (n = 652) using homologous protein profiles to investigate the potential for osmolyte metabolisms. Using the pattern of gene presence and absence, we infer the metabolic potential of surveyed microbes to interact with each osmolyte. Specifically, we identify: (1) complete pathways for osmolyte synthesis in both prokaryotic and eukaryotic marine microbes, (2) microbes capable of transporting osmolytes but lacking complete synthesis and/or breakdown pathways, and (3) osmolytes whose synthesis and/or breakdown appears to be specialized and is limited to a subset of organisms. The analysis clearly demonstrates that the marine microbial loop has the genetic potential to actively recycle osmolytes and that this abundant group of small metabolites may function as a significant source of nutrients through exchange among diverse microbial groups that significantly contribute to the cycling of labile carbon.
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